Abnormal gene product identified in hereditary dentatorubral-pallidoluysian atrophy (DRPLA) brain.

Dentatorubral-pallidoluysian atrophy (DRPLA) is associated with the expansion of an unstable CAG repeat. Using antibodies against a synthetic peptide corresponding to the sequence of the DRPLA gene product C terminus, we have identified the DRPLA gene product in normal human brains as a approximately 190 kD protein. We also find a larger approximately 205 kD protein specifically in DRPLA brains. Immunohistochemically, the DRPLA gene product is observed mainly in the neuronal cytoplasm. Our results demonstrate the existence of the expanded CAG repeat gene product and support the possibility that the expanded CAG-encoded polyglutamine stretch may participate in the pathological process of the similar trinucleotide repeat diseases.

Effects of ouabain on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats and in decerebrate and arterially perfused in situ preparation from juvenile rats.

The significance of Na/K-ATPase on respiratory rhythm generation is not well understood. We investigated the effects of the Na/K-ATPase blocker, ouabain, on respiratory rhythm. Experiments were performed with brainstem-spinal cord preparation from 0 to 3-day-old Wistar rats and with decerebrate and arterially perfused in situ preparation from juvenile rats (postnatal day 11-13). Newborn rat preparations were superfused at a rate of 3.0 ml/min with artificial cerebrospinal fluid, equilibrated with 95% O2 and 5% CO2, pH 7.4, at 26-27 °C. Inspiratory activity was monitored from the fourth cervical ventral root (C4). Application of ouabain (15-20 min) resulted in a dose-dependent increase in the burst rate of C4 inspiratory activity. After washout, the burst rate further increased to reach quasi-maximum values under each condition (e.g. 183% of control in 1 µM, 253% in 10 µM, and 303% in 20 µM at 30 min washout). Inspiratory or pre-inspiratory neurons in the rostral ventrolateral medulla were depolarized. We obtained similar results (i.e. increased phrenic burst rate) in an in situ perfused preparation of juvenile rats. Genes encoding the Na/K-ATPase α subunit were expressed in the region of the parafacial respiratory group (pFRG) in neonatal rats, suggesting that cells (neurons and/or glias) in the pFRG were one of the targets of ouabain. We concluded that Na/K-ATPase activity could be an important factor in respiratory rhythm modulation.

Dentatorubral-pallidoluysian atrophy proteins in lymphoblastoid cells.

The genetic defect responsible for dentatorubral-pallidoluysian atrophy (DRPLA) is an expansion of a CAG trinucleotide repeat. The DRPLA gene is translated into protein in the brain. In this study, we demonstrate that the wild-type and mutant genes are also translated into proteins, p190 and p205, in lymphoblastoid cells. The correlation between the age of onset and the expansion of polyglutamine stretch shown by slower electrophoretic mobility suggests that the polyglutamine stretch is directly involved in the acceleration of the disease process. Moreover, analysis of the protein in lymphoblastoid cells can be used as a diagnostic procedure for DRPLA.

Optical mapping reveals the functional organization of the trigeminal nuclei in the chick embryo.

The functional organization of the trigeminal nuclei during embryogenesis was investigated using multiple-site optical recording with a fast voltage-sensitive dye. Brainstem preparations with three classified trigeminal nerve afferents, the ophthalmic, maxillary and mandibular nerves, together with motor nerve fibers, were dissected from five- to eight-day-old chick embryos. Electrical responses evoked by trigeminal nerve stimulations were optically recorded simultaneously from many loci of the stained preparations. We identified three response areas related to the trigeminal nerve: area I, located cephalic to the level of the trigeminal ganglion; area II, located caudal to the level of the trigeminal ganglion; and area III, located at the level of the trigeminal root. The neural responses in areas I and II were evoked by ophthalmic, maxillary or mandibular nerve stimulation, while the responses in area III were detected when the stimulation was applied to the trigeminal motor nerve. In comparison with the morphology indicated by DiI labeling, the results suggest that areas I, II and III correspond to the principal sensory nucleus of the trigeminal nerve, the spinal sensory nucleus of the trigeminal nerve and the trigeminal motor nucleus, respectively. We identified two components of the optical response: a fast and a slow signal. In five-day-old preparations, fast spike-like signals related to action potentials were recorded from the three response areas. In six-day-old preparations, slow optical signals which reflect glutamate-mediated excitatory postsynaptic potentials were detected from area II only when the ophthalmic nerve was stimulated: no slow signal was evoked by maxillary or mandibular nerve stimulation. In seven- and eight-day-old preparations, slow signals were detected from both areas I and II with every nerve stimulation. These results suggest that synaptic function is first generated in the spinal trigeminal nucleus by the six-day embryonic stage, and the developmental organization of synaptic function is not the same in the three trigeminal nerves or in the two sensory nuclei. Contour line maps of the signal amplitude revealed that the size and the area of the neural responses within the trigeminal nuclei changed dramatically with development. We compared the spatial distribution and temporal dynamics of the optical signals between the ophthalmic, maxillary and mandibular nerve stimulations, and we found that somatotopic organization is less clear in a rostrocaudal/mediolateral X-Y plane, although the areas of the maxillary and mandibular nerves appeared to separate in the lateral direction.

Spreading depolarization waves triggered by vagal stimulation in the embryonic chick brain: optical evidence for intercellular communication in the developing central nervous system.

Throughout experiments on multiple-site voltage-sensitive dye recordings of neural activity in embryonic chick brain preparations, we have found a novel type of depolarization waves which spread widely from the brainstem to the whole brain region at a rapid rate (mm/s). This depolarization wave was triggered by glutamate-mediated postsynaptic potentials and was especially correlated to N-methyl-D-aspartate receptor function. Evidence that the spreading depolarization wave is eliminated by octanol or 18beta-glycyrrhetinic acid suggests that the depolarization wave depends on functions of gap junctions. The profile obtained with Ca(2+)-imaging experiments also suggests that the propagation of the depolarization wave is accompanied by a calcium wave. These results provide new evidence for intercellular functional communication between neural cells in the vertebrate central nervous system during embryonic development.

Optical responses to micro-application of GABA agonists in the embryonic chick brain stem.

We investigated optical responses induced by micro-application of muscimol and baclofen in the embryonic chick brain stem. Muscimol evoked biphasic optical signals which were similar to those induced by GABA. The first component was a dye-dependent absorption change that reflected membrane depolarization, and the second component was an intrinsic optical change coupled with changes in the membrane potential. On the other hand, baclofen did not elicit any optical change. The optical responses induced by muscimol persisted in the presence of picrotoxin and 2-hydroxysaclofen, suggesting that they contain a component which is not mediated by classical GABA receptors.

Effects of anisomycin on LTP in the hippocampal CA1: long-term analysis using optical recording.

Long-term potentiation (LTP) in the hippocampal CA1 region and in the dentate gyrus consists of different stages: early LTP lasting minutes or several hours, and late LTP lasting longer than 4 h. It has been suggested that the late phase of LTP is dependent on protein synthesis. However, the experimental results of the effects of protein synthesis inhibitors are still confusing. We applied optical recording techniques to rat hippocampal slices, and re-evaluated the effects of a protein synthesis inhibitor, anisomycin, on LTP. Using a voltage-sensitive oxonol dye, NK3630 (RH482), LTP in the CA1 region could be monitored optically for a long-term period (7-8 h). In the presence of anisomycin, the potentiation of the EPSP (excitatory postsynaptic potential) lasted about 2-3 h, followed by a gradual decline in the signal amplitude. Statistically, significant effects of anisomycin were observed 6 h after LTP induction for 100 Hz tetanus and 8 h after LTP induction for 400 Hz tetanus. These results suggest that the early phase of LTP is independent of protein synthesis, while the late phase of potentiation (> 3-5 h) depends on protein synthesis.

Optical detection of embryogenetic expression of vagal excitability in the rat brain stem.

We traced and identified the ontogenetic expression of neural excitability related to the vagus nerve in the embryonic rat brain stem. Multiple-site optical recordings of neural activities revealed two response areas in the E12 rat brain stem: one corresponding to the dorsal motor nucleus of the vagus nerve, and the other reflecting the activities of sensory nerve fibers. In embryos younger than E11, no optical response was identified, suggesting that excitability of the motoneurons and/or sensory nerve fibers is first generated no later than E12. A contour line map of the neural responses suggested that, in contrast to older embryos, the functional organization of the vagal nucleus is not orderly at the time of the initial expression of neural excitability.

Consistency behind trial-to-trial variation in intrinsic optical responses to single-whisker movement in the rat D1-barrel cortex.

We examined consistent characteristics behind the trial-to-trial variati on in intrinsic optical imaging of single barrel cortical responses to D 1-whisker movement in 2-5-week postnatal (2-5 W) and adul t (>9-weeks) Wistar rats, and we identified the effective are a of the neural response. The extent/size, configuration and orientation of the intrinsic optical response area varied from trial-to-trial with the same whisker stimulation. We argue that the trial-to-trial variation was due to cortical blood circulation related to the barrel neural activity. Subsequently, interpolating a family of the traces of the optical response area imaged with repeated stimulation for each animal, we extracted a centered circular area from the trial-to-trial response for each animal. Although the trial-to-trial variation decreased gradually with age, the spatial extent of the interpolated response area was consistently about 660 microm in diameter, in agreement with that measured morphologically and/or histochemically. A possible interpretation is that the optically defined area appears to image the actual effective single-barrel response area, as a first approximation. Furthermore, the constancy of the extracted area independent of age suggests that the barrel cortex is, in fact, virtually mature by 2 weeks of age. The extracted area was also nearly independent of the frequency (>/=5 Hz) of whisker movement.

Characterization of dentatorubral-pallidoluysian atrophy proteins using two-dimensional electrophoretic analysis.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is known to be an expansion of a CAG trinucleotide repeat. The mutant gene has been demonstrated to be translated into protein, and this protein contains an expanded polyglutamine region. In the present study, we have demonstrated using two-dimensional gel electrophoresis and immunoblotting that the DRPLA gene products consist of a number of isoelectric variants in brain tissue and lymphoblastoid cells. The identification of isoelectric variants indicates that there may be multiple stages of post-translational modification. DRPLA proteins from brain tissue possess fewer basic isoelectric variants than those from lymphoblastoid cells, suggesting that there is different post-translational modification steps in brain tissue and lymphoblastoid cell.

Expression of dentatorubral-pallidoluysian atrophy (DRPLA) proteins in patients.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a CAG trinucleotide repeat. The mutant gene is translated into protein whose electrophoretic mobility correlates to the number of expanded CAG trinucleotide repeats, indicating that the protein carries an expanded glutamine repeat. Using two polyclonal antibodies raised against the DRPLA gene product in immunoblotting, we determined the untruncated DRPLA proteins, and showed that the amounts of mutant and wild-type DRPLA proteins were similar in DRPLA brain tissues and lymphoblastoid cells, suggesting that regulation of the level of translation of the DRPLA gene is not central to the development of the disease.

Long tract degeneration in familial sudanophilic leukodystrophy with prominent spheroids.

We describe a family with an autosomal dominant neurodegenerative disorder, three siblings of which were verified by autopsy as having sudanophilic leukodystrophy (SLD). The clinical picture of the family is one of progressive dementia and spastic paralysis. Pathological examinations detected diffuse and patchy white matter lesions and the widespread presence of axonal spheroids in the lesions of all three autopsy patients. Because change was found selectively in the pyramidal tracts and optic radiations, this disease is considered to affect the long tracts systematically. The SLD in the family we studied is distinguished from other leukodystrophies by its clinical and pathological features, indicative that it may be a special type of SLD.

Mechanism of short-term memory and repetition in conduction aphasia and related cognitive disorders: a neuropsychological, audiological and neuroimaging study.

To evaluate the role of the sub-cortical white matter and cortical areas of the supramarginal gyrus in short-term memory impairment (shortened digit or letter span) and repetition difficulty, four patients with conduction aphasia and impaired short-term memory and two patients with only short-term memory impairment were given digit span, letter span, speech audiometry and dichotic listening tests. The results showed that in most of the patients letter span was inferior to digit span and that bilateral ear suppression in the dichotic listening test was observed in two patients with a lesion in the inferior part of the supramarginal gyrus, suggesting that what was affected was phonological information and that the supramarginal gyrus was the storage site. The overlapped lesion of conduction aphasia patients with short-term memory impairment was the periventricular white matter at the upper to middle part of the trigone, while patients with only short-term memory impairment had a lesion in the inferior supramarginal gyrus in common. Thus, damage to the periventricular white matter at the trigone may yield the phonemic paraphasia characteristic of conduction aphasia, while damage to the inferior part of the supramarginal gyrus may result in the impairment of short-term memory. We believe that as a part of the mechanisms of short-term memory and repetition, phonological information is processed in the primary auditory cortex and goes through the periventricular white matter to the inferior part of the supramarginal gyrus and is temporarily stored there.

[Gastric acid secretion and absorption of levodopa in patients with Parkinson's disease--the effect of supplement therapy to gastric acid].

Since an oral regimen of levodopa has been instituted for treatment of Parkinson's disease, its absorption and metabolism has been well demonstrated. However, its chemical characteristics of high solubility in acid solution and low solubility in water have not been well known. We paid attention to this characteristic and studied the relationship between its absorption and gastric acid secretion in 38 patients with Parkinson's disease who became refractory to therapy of levodopa. We measured the pH and amount of collected fasting gastric juice. Gastric acid secretion was decreased in 22 patients (58%). In ten of these 22 patients, 30 ml of lemon juice was prescribed in every administration of levodopa as a supplement to gastric acid for two weeks. Increases of L-dopa concentration after 60 min. and 180 min. were observed after lemon juice supplement therapy. Among the Parkinson symptoms, rigidity, akinesia, and small step gait were improved in every case except one patient who showed decrease of L-dopa concentration at 180 minutes. However, improvement of tremor was less remarkable. We consider this supplement therapy to gastric acid is one of the effective and useful methods in the management of Parkinson's disease.

Unique characteristics of ubiquitin-bonded complex play a pathological role in dentatorubral-pallidoluysian atrophy.

Abnormal complex formation of dentatorubral-pallidoluysian atrophy (DRPLA) protein and pathological ubiquitination of abnormal complex are two pathological processes involved in DRPLA neurodegeneration. Pathological ubiquitination and solubility in SDS and reducing agent are two unique characteristics of the DRPLA protein complex. Ubiquitination of abnormal DRPLA protein complex in DRPLA brain tissue is heat-resistant and stronger than that in control brain tissue. Pathological ubiquitination of DRPLA protein complex correlates with the onset of symptoms and the size of an expanded glutamine repeat in brain tissue of patients with DRPLA. Pathological ubiquitination plays an important role in DRPLA pathology. DRPLA protein complex is water-insoluble but soluble in SDS and reducing agent, and displays no difference in water insolubility between control and DRPLA brain tissue. Abnormal insoluble complex formation is not developed by a qualitative change in water insolubility of DRPLA protein complex but is developed by a spontaneous accumulation of an abnormally large amount of the DRPLA protein complex.

Aberrant phosphorylation of dentatorubral-pallidoluysian atrophy (DRPLA) protein complex in brain tissue.

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by DRPLA protein which carries expansion of a glutamine repeat. Abnormal high-molecular-mass complex formation by DRPLA protein and its pathological ubiquitination comprise the disease processes in the brains of patients with DRPLA. In this study, DRPLA protein complex was isolated and shown to have pathologically stronger bond formation with DRPLA proteins in DRPLA brain tissue compared with control brain tissue. Immunochemical methods and an enzymic dephosphorylation technique were used to demonstrate that DRPLA protein complex is aberrantly phosphorylated in DRPLA brain tissue. Immunohistochemical studies show that both the ubiquitinated cytoplasmic inclusions and the nuclear membrane are aberrantly phosphorylated in DRPLA-affected neurons. This finding suggests that the nuclear membrane is another pathological focus of DRPLA neurodegeneration.

Expanded glutamine repeat enhances complex formation of dentatorubral-pallidoluysian atrophy (DRPLA) protein in human brains.

The genetic defect in dentatorubral-pallidoluysian atrophy (DRPLA) is expansion of the CAG repeat. The mutant gene is translated into the protein which carries the expanded glutamine repeat. Immunoblots of human brain tissues with and without reduction show that the DRPLA protein is a disulfide-bond complex and that more of this complex is formed in DRPLA brains than in control brains. This suggests that DRPLA protein undergoes greater complex formation in DRPLA brains and the expanded glutamine repeat may enhance complex formation of untruncated DRPLA protein in DRPLA brains. Immunohistochemical findings show that DRPLA protein is localized in the cytoplasm of the neuron, evidence that it undergoes rare disulfide bonding there.

Different complex formations of dentatorubral-pallidoluysian atrophy (DRPLA) protein in human and rat neurons.

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a glutamine repeat in DRPLA protein. Rat DRPLA protein, homologous in sequence to human DRPLA protein, was identified in rat brains. Immunoblots of human control and rat brain tissues with and without reduction show that human DRPLA protein forms more disulfide-bond complexes than rat DRPLA protein. An immunohistochemical study and a subcellular fractionation experiment show that human DRPLA protein produces complexes within the cytoplasm of neurons, whereas rat DRPLA protein rarely does. In spite of the close homology of their amino acid sequences, human and rat DRPLA protein have different protein characters. The differences in these characters suggest structural differences and may be related to these proteins functions.

Abnormal dentatorubral-pallidoluysian atrophy (DRPLA) protein complex is pathologically ubiquitinated in DRPLA brains.

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a glutamine repeat in DRPLA protein. DRPLA protein undergoes greater complex formation in DRPLA brain tissue, and expanded glutamine repeat enhances complex formation of DRPLA protein. Immunoblots with and without reduction show that the DRPLA protein complex is ubiquitinated only in DRPLA brain tissue. Moreover, immunoblots of regional DRPLA brain tissues reveal that pathological ubiquitination of DRPLA protein complex is found selectively in affected lesions. Double-labeling immunohistochemical studies with antibodies against DRPLA protein and ubiquitin demonstrate that the DRPLA protein is co-localized with ubiquitin in DRPLA neurons and show characteristic neuronal cytoplasmic inclusions with ubiquitinated DRPLA protein complex in the center. Our findings suggest that DRPLA protein undergoes abnormal complex formation with expanded glutamine repeat, and then the complex is pathologically ubiquitinated in DRPLA brain tissue. Pathological ubiquitination of abnormal DRPLA protein complex plays a role in DRPLA pathology.

Food residue at endoscopy in patients who have previously undergone distal gastrectomy: risk factors and patient preparation.

BACKGROUND AND STUDY AIMS: Food residue is often seen in the gastric remnant after partial gastrectomy, making it difficult to diagnose early cancer in the residual stomach. The aims of this study were to clarify the risk factors for the accumulation of food residue, and to study methods of preparation for endoscopy in patients who had undergone distal gastrectomy. PATIENTS AND METHODS: 374 endoscopic examinations of patients who had undergone distal gastrectomy for gastric cancer were compared with 2168 endoscopic examinations in patients without a history of gastrectomy. Relationships between the presence of food residue and a number of clinical factors, including patient preparation, were evaluated by univariate and multivariate analyses. RESULTS: Food residue in the gastric remnant was observed in 70 examinations (18.7 %), a significantly higher proportion than that found in control patients (0.3 %). From multivariate analysis, underlying diseases (endocrine, metabolic, or connective tissue disease), Billroth type I reconstruction, and postoperative gastric retention were found to be independent risk factors for the accumulation of food residue. Diet preparation (a liquid diet plus aclatonium napadisilate) significantly decreased the incidence of food residue. CONCLUSIONS: Our diet preparation method can be recommended as a preparation for upper gastrointestinal endoscopy in patients who have undergone distal gastrectomy, especially in patients with additional risk factors.